Antimicrobial peptides (AMP) form a first line of defense of the innate immune system and have a broad spectrum of microbicidal activity against a wide range of Gram-negative and Gram-positive bacteria, fungi, protozoa, and even enveloped viruses. Recently they became a matter of increasing interest because of their excellent potential in treating diseases which cannot be cured by conventional antibiotics due to antimicrobial resistance. AMPs either induce membrane damage that is a lethal event for target bacteria or bind to several targets in the cytoplasmic region of the bacteria. All the evidence indicates that the action of the AMPs does not involve stereospecific protein-receptor recognition, since the interactions of AMPs with their targets are generally considered to be nonspecific. Therefore, the character of AMP interaction with bacterial cell wall lipids, viral envelope, or native plasma cell membrane lipids largely determine their lytic potential. As part of our study, novel planar biomimetic membranes, both at air-water and solid-liquid interface will be developed. This will allow use of highly sensitive structural experimental techniques, which cannot be employed with vesicle systems nor with real cells. Furthermore, in addition to AMP we also plan to investigate membrane interactions of their synthetic peptoid mimics (ampetoids), which have an advantage of being protease-resistant, while showing high potency and selectivity as antimicrobial agents. In this highly interdisciplinary proposal we plan to use cutting edge synchrotron X-ray scattering techniques, which together with AFM and epifluorescence studies will yield near atomic resolution of peptide-lipid interaction. These data will be used to advance the understanding of AMP and ampetoids mode of action which can be used to develop rational design strategies for AMPs and antimicrobial peptide mimics to advance development of highly potent drugs that are effective even against multidrug resistant bacteria and viruses. Specific aims of this project are: (1) Examine the modes of interaction of ampetoids and natural AMPs with lipid monolayers representing an outer leaflet of red blood cell membranes and surface layer of bacterial cell wall using synchrotron grazing incidence X-ray diffraction, X-ray reflectivity, epifluorescence microscopy, and AFM used in complementary manner. (2) Design novel fluid bilayer membranes at the air-water interface, use them to examine the interaction of AMPs and ampetoids with both leaflets of bilayer membrane. (3) Design novel cholesterol tethered bilayer lipid membranes (tBLM) with cytoskeleton component. Examine mechanism of AMPs and ampetoids interaction with these tBLMs and elucidate role of cytoskeleton in their interactions. The broader impact of the proposed research is to advance development of novel antibiotic and antiviral drugs that will be immune to bacterial and viral mutations. Antimicrobial peptides and their synthetic mimics have enormous potential with regard to bacterial resistance because they interact not only with specific membrane protein receptors, but also with the lipid matrix of cell membranes, whose lipid composition is highly unlikely to change as a result of bacterial mutation. Better understanding of antimicrobial peptides and peptoids mode of action on molecular level could enhance the design and development of potent alternatives to the conventional antibiotics and antiviral drugs used today.